Escribed by a bending modulus k, and lateral membrane compression/expansion within the bilayer plane, quantified as a lateral compression modulus Ks [43]. Membrane elasticity depends on the composition of the bilayer and its thermodynamic state (gel/liquid) [44]. In pure lipid membranes, it reflects interactions within the bilayer (lipid:lipid), the details of which are modified by the presence of any other molecules within, or along the aqueous interface of, the bilayer [40]. Thus, membrane elasticity responds to the insertion/adsorption of foreign molecules, in turn potentially affecting the association affinities of these molecules [45,46] or their function within the membrane [47,48,49]. We investigated the interaction and its mechanical effects, by developing a minimal giant unilamellar vesicle (GUV) model membrane system consisting of DOPC and DOPC-CL (95/5; mol/mol). This model, although highly simplified, may be considered to mimic mitochondrial contact sites. The micropipette aspiration technique was used to explore the order 58-49-1 effects of the presence of CL on the mechanical properties of the DOPC host membrane and of the apoptotic proteins tBid and caspase-8. The area expansion modulus (Ks), and the lysis tension (or tensile breaking strength, tr) were used to quantify membrane stability. The principal set-up and some results, compiled into two histograms, are shown in Fig. 3. The simple presence of any ofFlow Cytometry Analysis of the Functional Activity of the Caspase-8/Bid/cardiolipin PlatformFlow cytometry has rarely been used to follow and characterise giant unilamellar vesicles interacting with proteins [50,51]. We recently reported a flow cytometry analysis of giant unilamellar vesicles based on both their light scattering and fluorescence properties [41]. DprE1-IN-2 site CL-GUVs were analysed after the addition of protein and their fluorescence was recorded during the initial stages of the interaction (Fig. 5a). In the presence of caspase-8, vesicle fluorescence was detected immediately after the first addition of Bid (Fig. 5a). The subsequent addition of larger amounts of Bid-Alexa488 enhanced this fluorescence. In the absence of caspase-8, the increase in fluorescence was not significant (Fig. 5b). However, membrane-associated Bid fluorescence increased immediately after the addition of caspase-8 to the system.The Mitosome: Cardiolipin-Caspase-8-Bid(Fig. 4), the 15857111 binding of caspase-8 and Bid resulted in the disruption of CL-containing GUVs to form aggregates and smaller vesicles. In the absence of caspase-8, no change in side scatter was recorded (Fig. 5c, lower histogram): the initially injected vesicles were stable. We followed changes in the number of Bid-labelled vesicles over time (Fig. 5d). After the addition of caspase-8, all CL-containing GUVs were labelled with fluorescent Bid (closed circles). The number of Bid-labelled vesicles decreased over time, due to the cleavage of the Alexa488-labelled Bid domain after caspase-8 action. The large decrease in vesicle fluorescence provides further evidence for the activity of the newly formed caspase-8-Bid-CL platforms. Pre-incubation of the system with general caspase inhibitors (z-VAD-fmk and Boc-D-fmk) or a specific caspase-8 inhibitor (z-IETD-fmk) abolished the fluorescence drop due to Bid cleavage by caspase-8 (not shown).DiscussionCaspase-8 interacts with mitochondria in both healthy [52] and apoptotic [53,54] cells. However, it has remained unclear how caspase-8 interacts with CL in mi.Escribed by a bending modulus k, and lateral membrane compression/expansion within the bilayer plane, quantified as a lateral compression modulus Ks [43]. Membrane elasticity depends on the composition of the bilayer and its thermodynamic state (gel/liquid) [44]. In pure lipid membranes, it reflects interactions within the bilayer (lipid:lipid), the details of which are modified by the presence of any other molecules within, or along the aqueous interface of, the bilayer [40]. Thus, membrane elasticity responds to the insertion/adsorption of foreign molecules, in turn potentially affecting the association affinities of these molecules [45,46] or their function within the membrane [47,48,49]. We investigated the interaction and its mechanical effects, by developing a minimal giant unilamellar vesicle (GUV) model membrane system consisting of DOPC and DOPC-CL (95/5; mol/mol). This model, although highly simplified, may be considered to mimic mitochondrial contact sites. The micropipette aspiration technique was used to explore the effects of the presence of CL on the mechanical properties of the DOPC host membrane and of the apoptotic proteins tBid and caspase-8. The area expansion modulus (Ks), and the lysis tension (or tensile breaking strength, tr) were used to quantify membrane stability. The principal set-up and some results, compiled into two histograms, are shown in Fig. 3. The simple presence of any ofFlow Cytometry Analysis of the Functional Activity of the Caspase-8/Bid/cardiolipin PlatformFlow cytometry has rarely been used to follow and characterise giant unilamellar vesicles interacting with proteins [50,51]. We recently reported a flow cytometry analysis of giant unilamellar vesicles based on both their light scattering and fluorescence properties [41]. CL-GUVs were analysed after the addition of protein and their fluorescence was recorded during the initial stages of the interaction (Fig. 5a). In the presence of caspase-8, vesicle fluorescence was detected immediately after the first addition of Bid (Fig. 5a). The subsequent addition of larger amounts of Bid-Alexa488 enhanced this fluorescence. In the absence of caspase-8, the increase in fluorescence was not significant (Fig. 5b). However, membrane-associated Bid fluorescence increased immediately after the addition of caspase-8 to the system.The Mitosome: Cardiolipin-Caspase-8-Bid(Fig. 4), the 15857111 binding of caspase-8 and Bid resulted in the disruption of CL-containing GUVs to form aggregates and smaller vesicles. In the absence of caspase-8, no change in side scatter was recorded (Fig. 5c, lower histogram): the initially injected vesicles were stable. We followed changes in the number of Bid-labelled vesicles over time (Fig. 5d). After the addition of caspase-8, all CL-containing GUVs were labelled with fluorescent Bid (closed circles). The number of Bid-labelled vesicles decreased over time, due to the cleavage of the Alexa488-labelled Bid domain after caspase-8 action. The large decrease in vesicle fluorescence provides further evidence for the activity of the newly formed caspase-8-Bid-CL platforms. Pre-incubation of the system with general caspase inhibitors (z-VAD-fmk and Boc-D-fmk) or a specific caspase-8 inhibitor (z-IETD-fmk) abolished the fluorescence drop due to Bid cleavage by caspase-8 (not shown).DiscussionCaspase-8 interacts with mitochondria in both healthy [52] and apoptotic [53,54] cells. However, it has remained unclear how caspase-8 interacts with CL in mi.